The long term objective of this grant proposal is to gain a greater understanding of the catalytic role of ribosomal RNA in protein synthesis. Particular functions have already been assigned to many regions of both 16S and 23S rRNAs in E. coli. The aim of this project is to define these regions in greater detail, deciphering both the higher order structure and function, by utilizing a combination of powerful genetic, biochemical, biophysical and phylogenetic techniques. The recent discovery of a conformational switch in 16S rRNA affecting the mRNA pathway in the decoding region demonstrates the remarkable, dynamic nature of the ribosome. This encourages one to look for additional evidence of interactive relationships between different regions of rRNA within the subunits and between the subunits. The primary approach will involve rRNA mutagenesis of a plasmid-borne rrnB operon. Different host strains and plasmid vectors are available to permit even lethal mutations to be expressed. The technique of second site suppressor mutagenesis (or accelerated evolution) will be utilized to identify essential nucleotides comprising functional motifs and uncover more distant contacts representative of dynamic interactions. Sites of covariation and base triples identified by phylogenetic analysis will be mutagenized to further define three-dimensional structures such as the peptidyl transferase region. It is anticipated that these studies will further our understanding of the functional role of rRNA during all steps of translation (decoding, peptide bond formation and translocation) as they reveal the complex nature of the higher order structure and the multitude of dynamic processes which together define protein synthesis.